This clinical study aims to evaluate the antimicrobial effectiveness and postoperative pain outcomes of photodynamic therapy using Toluidine Blue O as an adjunct to conventional root canal treatment in patients diagnosed with symptomatic irreversible pulpitis. Photodynamic therapy is a non-invasive light-activated disinfection method, and this study will compare its performance with standard endodontic treatment protocols. The results are expected to provide clinical evidence regarding its potential benefits in reducing intracanal bacterial load and improving postoperative patient comfort.

This randomized clinical study is designed to investigate the antimicrobial effectiveness and postoperative pain outcomes of photodynamic therapy (PDT) using toluidine blue O (TBO) as an adjunct to conventional root canal treatment in single-rooted teeth with necrotic pulps. The rationale for this research is based on the persistent difficulty of achieving complete microbial elimination from the root canal system using standard chemomechanical preparation alone. Toluidine blue O-mediated PDT has been proposed as a minimally invasive disinfection method capable of producing reactive oxygen species after activation with visible light, thereby enhancing microbial reduction beyond that achieved by irrigation protocols.

The study employs a standardized protocol in which intracanal microbiological sampling is conducted at four predefined stages to monitor changes in microbial load throughout treatment. All samples are stored and analyzed using real-time quantitative polymerase chain reaction (qPCR), which allows sensitive detection and quantification of bacterial DNA. This molecular approach offers advantages over culture-based methods by identifying uncultivable or low-abundance bacterial species, providing a more comprehensive evaluation of the antimicrobial effect of PDT.

In the experimental group, photodynamic therapy is performed by introducing a 0.1 mg/mL toluidine blue O solution into the prepared root canal, followed by activation with a 630-nm LED light source. The choice of this wavelength corresponds to the absorption peak of TBO and optimizes energy transfer for singlet oxygen production. A sham procedure is used in the control group to maintain participant masking and ensure methodological consistency. Both groups receive identical chemomechanical preparation and irrigation procedures prior to the assigned intervention.

Pain assessment during the postoperative period is performed using a validated visual analog scale (VAS), providing continuous measurement of patient-reported discomfort during the first week after treatment. The integration of molecular microbiology with clinical pain evaluation allows a combined assessment of both biological and patient-centered outcomes. This study is expected to contribute new in-vivo evidence on the potential benefits of toluidine blue O-based PDT as an adjunctive disinfection method in endodontic therapy.

Inclusion Criteria:

1. Patients aged between 18 and 65 years
2. Presence of asymptomatic apical periodontitis with necrotic pulp in single-rooted maxillary or mandibular incisors, canines, or premolars
3. Periapical Index (PAI) score ≥ 2
4. Patients classified as ASA I (American Society of Anesthesiologists Physical Status Classification)
5. Preoperative pain score < 50 on the Visual Analog Scale (VAS)

Exclusion Criteria:

1. Multi-rooted teeth
2. Patients classified as ASA II or higher
3. Pregnant women or those with suspected pregnancy
4. Root canal curvature greater than 25° according to the Schilder method
5. Patients diagnosed with generalized periodontitis
6. Periodontal pocket depth > 3 mm in the related tooth
7. Use of analgesic medication within the past 72 hours
8. Use of antibiotic therapy within the past month
9. Presence of swelling and positive response to palpation/percussion tests

A single-blind design was implemented. Participants were unaware of their assigned intervention, and outcome assessors performing the microbiological analysis were masked to group allocation. A sham light application was used in the control group to preserve blinding